Hopane glycolipid, isolated from Bacillus acidocaldarius, was studied in its ability to support growth from Mycoplasma mycoides var. capri as substitute for sterols. All attempts to culture Mycoplasma on hopane glycolipid failed. When added together with cholesterol, the effect of HGL was a reduction in growth rate. The hopanoid diplopterol moderately supported growth of Mycoplasma. This effect was strikingly enhanced in diplopterol adapted cultures. Application of diplopterol via dipalmitoyl-phosphatidylcholine liposomes resulted in an improved growth rate. These results suggested that diplopterol can fulfill sterol function in Mycoplasma membranes.